CN109153954A

CN109153954A - Automated production and collection

Info

Publication number: CN109153954A
Application number: CN201780027623.XA
Authority: CN
Inventors: 波阿·旺; 布莱恩·J·南柯维斯
Original assignee: Terumo BCT Inc
Current assignee: Terumo BCT Inc
Priority date: 2016-05-05
Filing date: 2017-05-05
Publication date: 2019-01-04
Also published as: AU2022279399A1; WO2017193075A1; AU2017261348B2; JP2023182712A; EP3452575A1; EP3452575A4; JP2019514402A; JP2022033830A; AU2017261348A1; US20190382709A1; JP6986031B2

Abstract

The embodiments described herein provides generation, separation and/or the collection of the cellular products for discharging or secreting from cell.Cell can expand in (or extracapillary) space in the capillary of the bioreactor of the cell expansion system with culture medium.Cellular products can be discharged into the fluid space of bioreactor by cell.The example of the cellular products of this release includes extracellular particle (such as extracellular vesica (EV)).In order to collect the extracellular particle discharged from the cell being amplified, any extracellular particle from other sources is excluded, before collecting the extracellular particle discharged from amplifying cells, cleaning procedure can be used to eliminate any haemocyanin.The cellular products of release can be collected or are concentrated by control outlet parameter, and nutrients can reach cell for example, by the semi-permeable membrane that diffuses through of culture medium.Then the cellular products of release can be harvested.

Description

Automated production and collection

Cross reference to related applications

This application claims the U.S. Provisional Application Serial No. No.62/332 submitted on May 5th, 2016,426, and it is entitled " automated production and collection "；The U.S. Provisional Application Serial No. No.62/333 submitted on May 6th, 2016,013, and it is entitled " automated production and collection "；And the U.S. Provisional Application Serial No. No.62/500 that on May 3rd, 2017 submits, 962, and The priority and right of entitled " automated production and collection ".The disclosure of above-mentioned application passes through reference herein and is integrally incorporated this Text, as completely illustrated all the elements that it is instructed and for all purposes herein.

Background technique

Cell expansion system (CES) is for amplification and noble cells.Cell expansion system can be used for expanding (such as growth) Various attached cells and suspension cell.For example, cell expansion system can be used for amplification of mesenchymal stem cells (MSC) and other types Cell (such as bone marrow cell).The stem cell expanded from donorcells can be used for repairing or replacing impaired or defect tissue, And there is extensive clinical application in different diseases for many.Adherency and non-adhering type cell can be expanded in cell It is grown in bioreactor in system.

Summary of the invention

Embodiment of the present invention relates generally to generation, separation and/or collects the cell for discharging or secreting from cell and produces Object.The cellular products of this release or secretion can be described as releaser or secretion (released or secreted agent (s)), release component or secretion component, cellular products, cell generate component, release particle or secretory granules, release molecule or divide Secrete molecule, extracellular particle, release protein or secretory protein, metastasis etc..The example of such extracellular particle includes But be not limited to extracellular vesica (EV), viral vectors etc..

Extracellular vesica (EV) can be generated by cell, and during cell culture, EV can be discharged into culture or expansion Increase in its fluid or culture medium (due to the presence of the important by-products generated in the amplification phase, commonly referred to as conditioned medium). EV includes such as excretion body and microcapsule bubble.EV contain for cell communications and the vital RNA of other important cell processes, DNA and protein.For example, EV can be separated for example from body fluid (such as serum, blood plasma, urine) and cell culture supernatant.

In embodiments, cell-cell communication plays an important role in cell biology.Cell and other cell communications Ability makes it possible to occur the complex mechanism of protein synthesis etc..For example, cell can in several ways (such as directly Cell-cell contact or the transfer for secreting molecule) it communicates with one another.EV (such as microcapsule bubble and excretion body), which has in mediated cell, to be led to News and the ability for promoting hereditary information transfer.Microcapsule bubble is the direct rudiment (direct buds) from plasma membrane, and is usually wrapped Containing the surface marker similar with the film of origin.It, can shape when simultaneously rudiment enters extracellular space for vesica inner body and plasma membrane fusion At excretion body.Due to the positive effect of microcapsule bubble and excretion body in hereditary information transfer, microcapsule bubble and excretion body can be used for controlling Treat application.For example, EV can serve as antigen presenting cell to stimulate immune response and microcapsule bubble that can shift and activate chemotactic Factor acceptor, so as to cause anti-apoptotic effect.

In embodiments, cell expansion system can be used for amplifying cells.This amplification can be by using biological respinse Device or cell growth chamber occur.In one embodiment, this bioreactor or cell growth chamber are including, for example, hollow fibre Tie up film.This hollow-fibre membrane may include space (IC) in the space and capillary extracapillary (EC).Cell expansion system can be with It expands various kinds of cell type (such as mescenchymal stem cell, cancer cell, T cell, fibroblast and sarcoblast).These cells EV can be discharged into the fluid space of bioreactor by each in type, may then pass through outlet pocket collection.It is raw The semipermeable hollow fibers of object reactor allow required nutrients (such as glucose) to reach cell and metabolic waste by diffusion (such as lactic acid) is by diffusing out system.Unless also needing to harvest cell, otherwise cell can be retained in the hair of doughnut On the inside of tubule, and EV can be concentrated in fluid space, and EV then can be harvested from system without harvesting cell.

Embodiment of the present invention further to release is collected from the cell constantly expanded cellular products (such as EV or viral vectors etc.) before using the removal of auto-flushing program for cultivating or the haemocyanin of amplifying cells.This flushing Program allows system before since the cell being amplified collects the cellular products discharged, by first from appointing for amplification What serum or other sources remove the cellular products (for example, EV or viral vectors etc.) of any release to purify the cell of release and produce Object.

Embodiment of the present invention further provides for making it possible to collect or be concentrated by using multi-compartment bioreactor The cellular products of release.For example, by control outlet parameter (such as by closing IC outlet valve (outlet EC is kept to open)), it can To increase concentration of the cellular products in the side IC of release, and nutrients (such as glucose) still is able to by the side EC culture medium Addition and diffuse through film and reach the cell of the side IC.This collection can for example continue for some time (such as about 24 (24) hour to about 72 (72) hours).In one embodiment, this collection continued for about 48 (48) hours. After making this concentration increase of cellular products of release, the cellular products of release can be harvested to harvest bag or other containers In.According to an embodiment, attached cell can retain in the bioreactor during such harvest, until possible Need to discharge and harvest such cell (if any).

Embodiment of the present invention is provided by using one or more sides being used together with cell expansion system Case or task realize this production and/or collection of the cellular products of release.These schemes or task may include pre-programmed Scheme or task.In other embodiments, such scheme or task may include client or the customized scheme of user or appoint Business.By user interface (UI) and graphic user interface (GUI) element, client or the customized scheme of user can be created or appointed Business.Task may include one or more steps.In other embodiments, can choose pre-programmed, default or with The task that other modes are previously saved.Also in other embodiments, this production and/or collection can be by one or more A manual approach or task are used together to realize with cell expansion system.

Listing the content of present invention is to provide some concepts in simplified form, wherein these concepts are in following tool It is further described in body embodiment.The content of present invention is not intended to the range for limiting theme claimed in any way. In addition to provided herein is those of other than, may include feature (including its equivalent and variant).

Detailed description of the invention

Embodiment of the present invention can be described by reference to attached drawing.In the accompanying drawings, identical digital representation is identical Project.

Figure 1A depicts an embodiment of cell expansion system (CES).

Figure 1B shows the front view of an embodiment of bioreactor, it is shown that passes through following for the bioreactor Endless path.

Fig. 1 C depicts embodiment according to the present invention, for during the operation of cell expansion system rotatably or Laterally move the rocking equipment of cell growth chamber.

Fig. 2 shows the cell expansion systems of the fluid transport device with pre-installation of embodiment according to the present invention Perspective view.

Fig. 3 depicts the perspective view of the shell of the cell expansion system of embodiment according to the present invention.

Fig. 4 shows the perspective view of the fluid transport device of the pre-installation of embodiment according to the present invention

Fig. 5 depicts the schematic diagram of the cell expansion system of embodiment according to the present invention.

Fig. 6 shows the schematic diagram of the cell expansion system of another embodiment according to the present invention.

Fig. 7 depicts embodiment according to the present invention, illustrates the process of the component for generating and/or collecting release Operation characteristic flow chart.

Fig. 8 shows the cellular products for generating and/or collecting release for describing embodiment according to the present invention The flow chart of the operation characteristic of process.

Fig. 9 depicts the fortune of the method for producing and/or collecting releaser of explanation embodiment according to the present invention The flow chart of row feature.

Figure 10 shows the exemplary processing system according to an embodiment of the invention, the cell expansion system that may be implemented.

Figure 11 is shown according to an embodiment of the invention, extracting protein from the culture medium in cell expansion system Example results.

Figure 12 is shown according to an embodiment of the invention, being generated the example results of EV using cell expansion system.

Figure 13 A is shown according to an embodiment of the invention, being generated the example results of EV using cell expansion system.

Figure 13 B is shown according to an embodiment of the invention, being generated the example results of EV using cell expansion system.

Figure 13 C is shown according to an embodiment of the invention, being generated the example results of EV using cell expansion system.

Figure 14 is shown according to an embodiment of the invention, being generated the example results of EV using cell expansion system.

Specific embodiment

Following specific embodiments provide the discussion with the exemplary implementation scheme with reference to attached drawing.Include herein Specific embodiment is not necessarily to be construed as limitation (limiting) or shrinkage limit (restricting) present invention.Further, although In description embodiments herein, specific language can be used to feature, movement and/or structure, but claim is unlimited In described feature, movement and/or structure.It will be understood by those skilled in the art that including other improved embodiments at this In the spirit and scope of invention.It is possible to further using any substitution or addition, including as independent embodiment, or It is listed in conjunction with any other embodiment described herein.

Embodiment of the present invention generally relates to that release is generated, separated and/or collected in cell expansion system The system and method for cellular products (such as extracellular vesica (EV), viral vectors etc.).Embodiment of the present invention further mentions For that for example can collect or be concentrated the cellular products of release by using multi-compartment bioreactor.

In embodiments, doughnut permeability of the membrane allows by cell being retained in capillary in the circuit (IC) And separate cell with other components, for example, shla molecule can freely enter the circuit extracapillary (EC), for example, from And eliminate additional isolation step.Using the culture medium for the side EC for being added to bioreactor (for example, glucose, lactic acid, ammonia Base acid, vitamin) can satisfy culture in cell metabolic demand.This culture medium can diffuse through bioreactor Semi-permeable membrane.Molecular weight is too big and cannot diffuse through the nutrient media components of film ultrafiltration can be used and (such as accomplished continuously or intermittently push away Note addition) it is added to the side IC of bioreactor.In one embodiment, ultrafiltration (closing of IC outlet valve) can be used to protect Stay any component too big and that the film on the side bioreactor IC cannot be diffused through.It may not be able to be expanded by the EV that cell generates It dissipates through film, i.e. its molecular weight may be too big.Therefore, in the case that EV concentration can be continuously increased, EV can amplification (or The collection phase of restriction) during be maintained at the side IC of bioreactor.It then can be for example at a prescribed interval or in whole process At the end of by EV from the side IC of bioreactor be collected into harvest container or harvest bag in.In two streams of not hollow-fibre membrane In the case where the benefit of body compartment, according to embodiment, EV concentration or collection can be limited to cell generate EV rate and can Fresh culture to be added to the rate for meeting nutritional need in culture environment.

For example, (such as pass through by control outlet parameter and close IC outlet valve (keep EC outlet open)), therefore can be with Increase the concentration in the cellular products of the release of the side IC, and nutrients (such as glucose) still is able to train by adding in the side EC It supports base and diffuses through film and reach the cell of the side IC.In other embodiments, nutrients can be cultivated by adding in the side IC Base supports the cell of the side IC.In a further embodiment, cell amplification can occur in the side EC, and the side IC (or The side EC) it adds culture medium and diffuses through film to reach cell.The collection of EV can be continued for a period of time (such as about 24 (24) hour or longer time).In other embodiments, this collection can continue for less than for about 24 (24) hours.? In other embodiments, this collection can continue for example, about 48 (48) hours to about 72 (72) hours.Make to release After this concentration increase for the cellular products put, the cellular products of release can be harvested into harvest bag or other containers.Root According to an embodiment, attached cell can retain during such harvest in the bioreactor, until needing to discharge With the such cell (if any) of harvest.

As described above, embodiment is related to cell expansion system.In embodiments, this cell expansion system is closing , wherein closing cell's amplification system includes the content for being not directly exposed to atmosphere.This cell expansion system can be certainly Dynamicization.In embodiments, adherent and non-non-adherent or suspension type cell can be in the biology in cell expansion system It is grown in reactor.According to embodiment, cell expansion system may include basal medium or other kinds of culture medium.It provides The method of the supplementing culture medium of cell growth for occurring in the bioreactor of closing cell's amplification system.Implementing In scheme, the bioreactor being used together with this system can be hollow-fiber bioreactor.Reality according to the present invention Scheme is applied, the bioreactor of many types can be used.

In embodiments, which may include bioreactor, which further comprises having at least phase The first fluid flow path of opposite end, the first opposite end of first fluid flow path and the first port fluid of hollow-fibre membrane The connected and second end of first fluid flow path and the second port fluid of hollow-fibre membrane are connected, wherein first fluid Flow path includes the capillary inner part of hollow-fibre membrane.In embodiments, hollow-fibre membrane includes multiple doughnuts. The system can further comprise the fluid inlet path being connected with first fluid flow path fluid, and plurality of cell passes through the One fluid inlet path introduces first fluid flow path.It can also include flowing road for the first fluid in bioreactor First pump of circulation of fluid in diameter.In embodiments, which includes the controller for controlling the operation of the first pump.One In a embodiment, controller is, for example, the computer system for including processor.In embodiments, controller is configured as controlling System pump in first fluid flow path with first rate circulation of fluid.In some embodiments, it including is used for capillary Entrance fluid culture medium bag out of capillary is transferred to the second pump of first fluid flow path and for controlling the second pump in managing Operation second controller.In embodiments, the second pump of second controller control, such as cell is turned from cell entry bag Move on to first fluid flow path.According to embodiment, additional controller, such as third controller, the 4th control can be used Device processed, the 5th controller, the 6th controller etc..Further, according to an embodiment of the invention, additional pump can be used, Such as third pump, the 4th pump, the 5th pump, the 6th pump etc..In addition, though the present invention can be related to culture medium bag, cell entry bag Deng, multiple bags (such as the first culture medium bag, the second culture medium bag, third culture medium bag, the first cell entry bag, the second culture Base, third cell entry bag etc.) and/or other kinds of container can use in embodiments.In other embodiments, Single culture medium bag, individual cells inlet pocket etc. can be used.Further, may include in embodiments it is additional or Other fluid paths (such as second fluid flows path, second fluid ingress path etc.).

In other embodiments, system for example states control by lower item: being coupled to the processor of cell expansion system；Display Device, and processor communication, and can operate to show data；With memory (memory), with processor communication and can be by handling Device is read, and includes series of instructions.In embodiments, when instruction is executed by processor, processor reception is for example coated The instruction of bioreactor.The instruction of response coating bioreactor, processor can execute series of steps to coat biology Reactor, and can next receive the instruction by loading cells into bioreactor.Response loads the instruction of cell, place Reason device can execute series of steps so that cell to be downloaded in bioreactor from such as cell entry is packed.

According to an embodiment of the invention, depicting the schematic diagram of exemplary cells amplification system (CES) in Figure 1A. " CES " and " system " may be used interchangeably.CES 10 includes first fluid circulating path 12 and second fluid circulating path 14.Root According to embodiment, first fluid flow path 16 has at least opposite end 18 and 20, they and doughnut cell are grown (referred to herein as " the bioreactor ") fluid of room 24 is connected.Specifically, opposed end 18 can be with cell growth chamber 24 22 fluid of first entrance be connected, and opposed end 20 can be connected with 28 fluid of first outlet of cell growth chamber 24.The Fluid in one circulating path 12 flows through the hollow fibre for the hollow-fibre membrane 117 (referring to Figure 1B) being arranged in cell growth chamber 24 Tie up the inside of 116 (referring to Figure 1B) (cell growth chamber and hollow-fibre membrane are described in more detail below).Further, One fluid flow control device 30, which can be operably connected, first fluid flow path 16 and can control first circulation road Fluid flowing in diameter 12.

Second fluid circulating path 14 includes second fluid flowing path 34, cell growth chamber 24 and second fluid flowing control Device 32 processed.According to embodiment, second fluid, which flows path 34, has at least opposite end 36 and 38.Second fluid flowing The opposed end 36 and 38 in path 34 can be connected with the ingress port 40 of cell growth chamber 24 and 42 fluid of outlet port respectively. The fluid for flowing through cell growth chamber 24 can be with the external contact of the hollow-fibre membrane 117 in cell growth chamber 24 (referring to figure 1B), wherein hollow-fibre membrane includes multiple doughnuts.Second fluid circulating path 14 can be operably connected second Body flow control apparatus 32.

Therefore, the first and second fluid circulation paths 12 and 14 can be by hollow-fibre membrane 117 in cell growth chamber 24 Middle separation (referring to Figure 1B).Fluid in first fluid circulating path 12 flows through the capillary of the doughnut in cell growth chamber 24 The space (" IC ") in pipe.First circulation path 12 can be referred to as " circuit IC ".Fluid in second circulation path 14 flows through carefully Space extracapillary (" EC ") in intracellular growth room 24, second fluid circulating path 14 are properly termed as " EC loop ".According to implementation Scheme, fluid in first fluid circulating path 12 can be relative to the fluids in second fluid circulating path 14 with cocurrent or right The mode of stream flows.

Fluid inlet path 44 can be connected with 12 fluid of first fluid circulating path.Fluid inlet path 44 allows fluid Into first fluid circulating path 12, and fluid outlet path 46 allows fluid to leave CES 10.Third fluid flowing control dress Setting 48 can be operably connected fluid inlet path 44.Alternatively, third fluid flow control device 48 alternatively connects One outlet pathway 46.

According to embodiment, as used herein fluid flow control device may include pump, valve, fixture or combinations thereof. Multiple pumps, valve and fixture can be arranged in any combination.In various embodiments, fluid flow control device is or including wriggling Pump.In embodiments, fluid circulation paths, ingress port and outlet port can be made of the pipeline of any material.

Various assemblies " being operably connected " used herein.As used herein, it " is operably connected " and refers to can grasp The component that the mode of work links together, and the embodiment being directly connected to including wherein component, and wherein add-on assemble The embodiment being placed between two chain joint assemblies." being operably connected " component can be " fluid is connected "." fluid phase Even " refer to the component to link together, fluid is transported between device." fluid is connected " sets including wherein additional component It sets between two fluid connected components, and the embodiment for the component being directly connected to.Fluid connected components may include not connecing It touches fluid but contacts other component with the component of steerable system (for example, fluid pumping is passed through flexibility by the outside by compressed pipe The peristaltic pump of pipe).

In general, any kind of fluid (including such as buffer, the fluid containing protein and fluid containing cell) can To flow through various circulating paths, ingress path and outlet pathway.As used herein, " fluid ", " culture medium " and " fluid culture Base " is used interchangeably.

Figure 1B is gone to, the doughnut cell growth chamber 100 that can be used with the present invention is shown with front elevational view Example.Cell growth chamber 100 has longitudinal axis L A-LA and including cell growth chamber shell 104.In at least one embodiment party In case, cell growth chamber shell 104 includes four openings or port: IC ingress port 108, IC outlet port 120, EC arrival end 128 and EC of mouth outlet port 132.

According to an embodiment of the invention, the fluid in first circulation path the first of cell growth chamber 100 by indulging Enter cell growth chamber 100 to the IC ingress port 108 of end 112, enters through in multiple including hollow-fibre membrane 117 On the inside of the capillary of hollow fiber 116 (referring in various embodiments, as the side (" IC ") in the capillary of hollow-fibre membrane or " space IC "), and cell life is left by the IC outlet port 120 for the second longitudinal direction end 124 for being located at cell growth chamber 100 Long room 100.Fluid path between IC ingress port 108 and IC outlet port 120 defines the part IC of cell growth chamber 100 126.Fluid in second circulation path is flowed into cell growth chamber 100 by EC ingress port 128, with doughnut 116 On the outside of capillary or external (referred to as " side EC " or " space EC " of film) contacts, and leaves cell life by EC outlet port 132 Long room 100.Fluid path between EC ingress port 128 and EC outlet port 132 includes the part EC of cell growth chamber 100 136.It can be with the external contact of doughnut 116 via the fluid that EC ingress port 128 enters cell growth chamber 100.Small point Sub (for example, ion, water, oxygen, lactic acid etc.) can be diffused by doughnut 116 from the inside of doughnut or the space IC outer Portion or the space EC, or from the space EC to the space IC.Macromolecule molecule (such as growth factor) is typically too big and cannot pass through Hollow-fibre membrane, and may be retained in the space IC of doughnut 116.In embodiments, training can be replaced as needed Support base.Culture medium can also be by oxygenator or gas shift module circulation with exchanging gas as needed.According to embodiment, Cell may be embodied in first circulation path and/or second circulation path as described below, and can film the side IC and/ Or on the side EC.

Material for manufacturing hollow-fibre membrane 117 can be any biocompatible polymer material, can be made Doughnut.According to an embodiment of the invention, a kind of material that can be used is the material based on polysulfones of synthesis.According to reality Scheme is applied, in order to make cell adherence on the surface of doughnut, surface can be changed in some way, it can be by with protein (such as fibronectin (FN) or collagen) at least coats cell growth surface, or under the radiation by exposed surface.γ is penetrated The attachment of the permission attached cell of the film surface of line processing, without in addition using the coating films such as fibronectin, cool insoluble protein.By γ Bioreactor made of the film of Irradiation may be reused.It can be used with embodiment according to the present invention and be used for cell Other coatings of attachment and/or processing.

In embodiments, CES (such as CES 500 (referring to Fig. 5) and/or CES 600 (for example, see Fig. 6)) may include It is configured to by the way that cell growth chamber is connected to rotation and/or side rocking device, other portions relative to cell expansion system It is moved on part or the device of " shake " cell growth chamber.Fig. 1 C shows a kind of such device, wherein according to an embodiment party Case, bioreactor 100 can be rotationally connected to two rotation teeter members and a side rocking component.

First rotation teeter member 138 rotates bioreactor 100 around the central axis 142 of bioreactor 100. Rotation teeter member 138 can rotatably be connected with bioreactor 100.In embodiments, bioreactor 100 can enclose Around the continuous rotation in a single direction in clockwise and counterclockwise directions of central axis 142.Alternatively, according to embodiment, biology Reactor 100 can for example be rotated around central axis 142 in an alternating manner, for example, rotating clockwise first, then counterclockwise Rotation.

CES can also include the second rotation teeter member, rotate bioreactor 100 around rotation axis 144.Rotation Shaft axis 144 can pass through the central point of bioreactor 100 and can be perpendicular to central axis 142.In embodiments, Bioreactor 100 can be around the continuous rotation in a single direction in clockwise and counterclockwise directions of rotation axis 144.Or Person, bioreactor 100 can for example be rotated in an alternating manner around the rotation of central axis 144, be rotated clockwise first, then Rotation counterclockwise.In various embodiments, bioreactor 100 can also around rotation axis 144 rotate and relative to Gravity is placed in horizontal or vertical direction.

In embodiments, side rocking component 140 can be with 100 side of bioreactor to being connected.In embodiments, The plane of side rocking component 140 transverse shifting on-x and the direction-y.It, can be by doughnut according to embodiment The movement of celliferous culture medium reduces the sedimentation of cell in bioreactor.

The rotation and/or lateral movement of rocking equipment can reduce the sedimentation of device inner cell and reduce in bioreactor A part in capture cell a possibility that.According to Stoke's law (Stoke ' s Law), cell settles in cell growth chamber Rate it is directly proportional to the density contrast between cell and suspension medium.In certain embodiments, repeatedly 180 degree as described above Rotation (quick) simultaneously suspends (such as with 30 seconds total assembly time) and makes non-adhesive red blood cell suspension.In an embodiment party In case, a minimum of about 180 degree is preferably rotated；However, it is possible to use up to 360 degree or more of rotation.The different components that wave can To be used alone, or can be combined with any combination.For example, waving around 142 curled curved surface 100 of central axis Component can be combined with the teeter member around 144 curled curved surface 100 of axis.It equally, can be only in any combination On the spot execute being rotated both clockwise and counterclockwise around not coaxial line.

Fig. 2 is gone to, embodiment according to the present invention shows the cell amplification of the fluid transport component with pre-installation The embodiment of system 200.CES 200 includes that cell expands machine 202, and cell amplification machine 202 includes for expanding machine with cell The hatch or closable door 204 that 202 rear portion 206 engages.Inner space 208 in cell amplification machine 202 includes being suitable for connecing Receive and engage the feature of the fluid transport component 210 of pre-installation.The fluid transport component 210 of pre-installation is detachably connected to carefully Born of the same parents expand machine 202, in order to promote the used pre-installation on same cell amplification machine 202 at cell amplification machine 202 The fluid transport component 210 of pre-installation that fluid transport component 210 relatively quickly more renews or not used.List can be operated Cell expands machine 202 to use the fluid transport component 210 of the first pre-installation and grows or expand first group of cell, and hereafter It can be for growing or expanding second group of cell using the second pre-installation fluid transport component 210, without pre- first It carries out disinfection between the fluid transport component 210 of installation and the replacement of fluid transport component 210 of the second pre-installation.The stream of pre-installation Body transport assembly 210 includes bioreactor 100 and oxygenator or gas transport module 212 (referring also to Fig. 4).According to embodiment party Case, pipeline guiding groove are shown as 214, and the various culture mediums of the fluid transport component 210 of pre-installation are connected to for reception pipe.

Next, Fig. 3 show embodiment according to the present invention in the fluid transport for removably adhering to pre-installation The rear portion 206 of cell amplification machine 202 before component 210 (Fig. 2).Door 204 can be closed by being omitted in Fig. 3 (shown in Fig. 2).Carefully The rear portion 206 that born of the same parents expand machine 202 includes many different structures, the element group for the fluid transport component 210 with pre-installation Close work.More specifically, the rear portion 206 of cell amplification machine 202 includes multiple fluid transport components 210 for pre-installation Pump loop cooperation peristaltic pump, including IC circulating pump 218, EC circulating pump 220, IC inlet pump 222 and EC inlet pump 224.This Outside, cell amplification machine 202 rear portion 206 include multiple valves, including IC circulating valve 226, reagent valve 228, IC culture medium valves 230, Air removes valve 232, cell entry valve 234, washing valve 236, distributing valve 238, EC culture medium valves 240, IC waist valve 242, EC Waist valve 244 and collection valve 246.Multiple sensors are also connected with the rear portion 206 of cell amplification machine 202, including IC outlet pressure Sensor 248, IC inlet pressure and temperature combination sensor 250, EC entrance combination pressure and temperature sensor 252 and EC Outlet pressure sensor 254.According to an embodiment, further it is shown that the optical sensor for air removal room (ARC) 256。

According to embodiment, the axis or rocking rod controller 258 for curled curved surface 100 are shown.With axis or shake The connected axis accessory 260 of rod controller 258 allows axis to pass through the suitable alignment in hole, (schemes see, for example, the 424 of pipeline organizer 4) 400 of the rear portion 206 of machine 202 together, are expanded see, for example, 300 (Fig. 4) of pre-installation transport assembly 210 or with cell.Axis Or the rotation of rocking rod controller 258 assigns axis accessory 260 and 100 rotary motion of bioreactor.Therefore, as the behaviour of CES 200 Author or user add new or not used preassembled fluid transport component 400 (Fig. 4) and expand machine 202 to cell, right Standard is relatively simple thing, and the axis for being properly oriented preassembled fluid transport component 210 or 400 passes through hole 424 (Fig. 4) Be furnished with axis accessory 260.

Fig. 4 is gone to, the perspective view of the fluid transport component 400 of detachably connected pre-installation is shown.Pre-installation Fluid transport component 400 can be detachably connected to cell amplification machine 202 (Fig. 2 and 3), in order to expand machine 202 in cell It is new that place relatively quickly will use preassembled fluid transport component 400 to be changed at same cell amplification machine 202 Or the fluid transport component 400 of not used pre-installation.As shown in figure 4, bioreactor 100 may be coupled to including axis accessory 402 bioreactor shaft joint.Axis accessory 402 includes one or more axis retention mechanisms, is such as expanded for zygoneure The bias arm or spring member 404 of the axis (such as 258 (being shown in Fig. 3)) of machine 202.

According to embodiment, the fluid transport component 400 of pre-installation includes pipeline 408A, 408B, 408C, 408D, 408E Deng and various pipe fittings, to provide fluid path as illustrated in Figures 5 and 6 as described below.Pump loop can also be provided for pump 406A and 406B.In embodiments, although various culture mediums can be provided at the position that cell expands 202 place of machine, It is according to embodiment, the fluid transport component 400 of pre-installation may include enough length of tube to extend to cell amplification machine 202 outside and the pipeline being connected with culture medium bag or container can be welded to.

Next, Fig. 5 shows the schematic diagram of the embodiment of cell expansion system 500, and Fig. 6 shows cell amplification The schematic diagram of another embodiment of system 600.As shown in the embodiment in Figures 5 and 6, and it is as described below, and cell exists IC is grown in space.However, the present invention is not limited to these Examples, and cell can be provided in other embodiments in EC It is grown in space.

According to embodiment, Fig. 5 shows CES 500 comprising 502 (also referred to as " capillary of first fluid circulating path Inner ring road " or " IC loop ") and second fluid circulating path 504 (also referred to as " extracapillary loop " or " EC loop ").First Fluid flow path 506 can be connected to form first fluid circulating path 502 with 501 fluid of cell growth chamber.Fluid passes through IC ingress port 501A flows into cell growth chamber 501, by the doughnut in cell growth chamber 501, and passes through the outlet end IC Mouth 501B leaves.Pressure gauge 510 measures the pressure for leaving the culture medium of cell growth chamber 501.Culture medium, which flows through, can be used for controlling The IC circulating pump 512 of culture medium flow velocity.IC circulating pump 512 can be pumped along first direction or the second direction opposite with first direction Send fluid.Outlet 501B may be used as reversed entrance.Culture medium into the circuit IC can be entered by valve 514.Such as ability Field technique personnel will be understood that, can place at various locations additional valve, pressure gauge, pressure/temperature sensor, port and/ Or other devices are to be isolated and/or measure the culture medium feature along the part of fluid path.Shown in it should therefore be understood that Schematic diagram indicates a kind of possible configuration of the various elements of CES 500, and shown in schematic diagram modification at one or more In the range of multiple existing embodiments.

About the circuit IC 502, media samples can be obtained from sample port 516 or sample coil 518 during operation. The Pressure/Temperature meter 520 being arranged in first fluid circulating path 502 allows to detect culture medium pressure and temperature during operation Degree.Then culture medium is back to IC ingress port 501A to complete fluid circulation paths 502.Grown in cell growth chamber 501/ The cell of amplification can be flushed in harvest bag 599 by valve 598 from cell growth chamber 501, or the weight in doughnut New distribution is with further growth.

Fluid in second fluid circulating path 504 enters cell growth chamber 501 via EC ingress port 501C, and via EC outlet port 501D leaves cell growth chamber 501.Culture medium in the circuit EC 504 can in cell growth chamber 501 in The external contact of hollow fiber, to allow small molecule diffusion disengaging doughnut.

According to an embodiment, the permission of Pressure/Temperature meter 524 being arranged in second fluid circulating path 504 is being trained The space EC that feeding base enters cell growth chamber 501 measures the pressure and temperature of culture medium before.Pressure gauge 526 allows in culture medium Leave the pressure for the culture medium that cell growth chamber 501 is measured later in second fluid circulating path 504.It, can be with about the circuit EC Media samples are obtained from sample port 530 or sample coil during operation.

In embodiments, after leaving the EC outlet port 501D of cell growth chamber 501, second fluid circulating path Fluid in 504 reaches oxygenator or gas transport module 532 by EC circulating pump 528.What EC circulating pump 528 may be reversed Direction pumps fluid.Second fluid flow path 522 can by oxygenator ingress port 534 and oxygenator outlet port 536 with Oxygenator or 532 fluid of gas transport module are connected.In operation, broth is flowed into via oxygenator ingress port 534 Oxygenator or gas transport module 532, and oxygenator or gas transport module are left by oxygenator outlet port 536.Example Such as, the culture medium of oxygenator or gas transport module 532 into CES 500 adds oxygen and therefrom removes bubble.In various realities It applies in scheme, the culture medium in second fluid circulating path 504 can be with the gas into oxygenator or gas transport module 532 In balance.Oxygenator or gas transport module 532 can be any suitably sized oxygenator or charge delivery mechanism.Air Or gas flows into oxygenator or gas transport module 532 by filter 538, and flows out oxygenator or gas by filter 540 Body transmitting device 532.Filter 538 and 540 reduces or prevents the dirt of oxygenator or gas transport module 532 and related culture medium Dye.Oxygenator can be passed through from the air or gas that CES 500 is purified during the part event (priming sequence) is perfused Or gas transport module 532 is discharged into atmosphere.

In the configuration described for CES 500, in first fluid circulating path 502 and second fluid circulating path 504 Broth cell growth chamber 501 is flowed through with the same direction (and stream configuration).CES 500 may be configured to convection current Structure flowing.

According at least one embodiment, the culture medium including cell (can be come via first fluid flow path 506 From bag 562) and broth from bag 546 introduce first fluid circulating path 502.Fluid container 562 is (for example, be used for By the cell entry bag or perfusion of saline liquid of air tapping system, air bleed system) it can be via valve 564 and first fluid flow path 506 and the One fluid circulation paths, 502 fluid is connected.

Fluid container or culture medium bag 544 (for example, reagent) and 546 (for example, IC culture mediums) can be respectively via 548 Hes of valve 550 with first fluid ingress path 542, or be connected via valve 570 and 576 with 574 fluid of second fluid ingress path.Also provide Paths 508 and 509 are perfused in first and second sterile salable inputs.Air removes room (ARC) 556 can be with first circulation road 502 fluid of diameter is connected.It may include one or more ultrasonic sensors that air, which removes room 556, which includes Upper sensor and lower sensor are to detect air, fluid shortage and/or air/fluid interface (for example, removing in air The gas/fluid interface at certain measurement positions in room 556).For example, can air remove room 556 bottom near and/ Or near top detects air, fluid and/or air/fluid interface at these positions using ultrasonic sensor.Not In the case where being detached from the spirit and scope of the present invention, embodiment provides the use of many other types of sensor.For example, Optical sensor can be used with embodiment according to the present invention.From CES during the part of perfusion event or other schemes The air or gas of 500 purifications can be discharged into the atmosphere outside air valve 560 via pipeline 558, and pipeline 558 can remove room with air 556 fluids are connected.

EC culture medium (for example, come from bag 568) or washing solution (for example, coming from bag 566) can be added to first or Second fluid flows in path.Fluid container 566 can be connected with 570 fluid of valve, and valve 570 can be via distributing valve 572 and One fluid inlet path 542 is connected with 502 fluid of first fluid circulating path.Alternatively, by opening valve 570 and closing distributing valve 572, fluid container 566 can be via second fluid ingress path 574 and EC ingress path 584 and second fluid circulating path 504 fluids are connected.Similarly, fluid container 568 can be connected with 576 fluid of valve, and valve 576 can be via first fluid entrance road Diameter 542 and distributing valve 572 are connected with 502 fluid of first fluid circulating path.Alternatively, by opening valve 576 and closing distribution Valve 572, fluid container 568 can be connected with 574 fluid of second fluid ingress path.

Optional heat exchanger 552 can be provided to introduce for culture medium reagent or washing solution.

In the circuit IC, fluid can be promoted initially by IC inlet pump 554.In the circuit EC, fluid can be initially by EC Inlet pump 578 promotes.The air monitor 580 of such as ultrasonic sensor can also be connected with EC ingress path 584.

In at least one embodiment, the first and second fluid circulation paths 502 and 504 are connected to waste line 588. When valve 590 is opened, IC culture medium can flow through waste line 588 and flow to litter bag or outlet pocket 586.Equally, work as valve When 582 opening, EC culture medium can flow to litter bag or outlet pocket 586 by waste line 588.

In embodiments, path 596 can be harvested by cell and harvests cell.It herein, can be by the way that cell will be contained IC culture medium path 596 and valve 598 harvested by cell be pumped to cell harvest bag 599 and harvest from cell growth chamber 501 cell.

The various parts of CES 500 may include or be contained in machine or shell (such as cell amplification machine 202 (Fig. 2 and 3)), wherein the machine for example maintains cell and culture medium at a predetermined temperature.

Go to Fig. 6, it is shown that the schematic diagram of another embodiment of cell expansion system 600.CES 600 includes first Fluid circulation paths 602 (also referred to as " capillary inner ring road " or " IC loop ") and second fluid circulating path 604 is (also referred to as " extracapillary loop " or " EC loop ").First fluid flow path 606 can be connected with 601 fluid of cell growth chamber with shape At first fluid circulating path 602.Fluid flows into cell growth chamber 601 by IC ingress port 601A, passes through cell growth chamber Doughnut in 601, and left by IC outlet port 601B.Cell growth chamber 601 is left in the measurement of pressure sensor 610 The pressure of culture medium.Other than pressure, in embodiments, sensor 610 is also possible to detect culture medium during operation The temperature sensor of pressure and temperature.Culture medium flows through the IC circulating pump 612 that can be used for controlling culture medium flow velocity.IC circulating pump 612 can pump fluid along first direction or the second direction opposite with first direction.Outlet 601B may be used as reversed entering Mouthful.Culture medium into the circuit IC can be entered by valve 614.As it will appreciated by a person of ordinary skill, can be each Additional valve, pressure gauge, pressure/temperature sensor, port and/or other devices are placed to be isolated and/or measure edge in position The culture medium feature of each section of fluid path.The various members of the expression of schematic diagram shown in it should therefore be understood that CES 600 A kind of possible configuration of part, and shown in schematic diagram modification in the range of one or more the present embodiment.

About the circuit IC, media samples can be obtained from sample coil 618 during operation.Then culture medium returns to IC ingress port 601A is to complete fluid circulation paths 602.Growth/amplification cell can pass through in cell growth chamber 601 Valve 698 and pipeline 697 are flushed in harvest bag 699 from cell growth chamber 601.Alternatively, after valve 698 is closed, Ke Yi Cell is redistributed in room 601 with further growth.

Fluid in second fluid circulating path 604 enters cell growth chamber 601 via EC ingress port 601C, and via EC outlet port 601D leaves cell growth chamber 601.According to an embodiment, the culture medium in the circuit EC can be raw with cell The external contact of doughnut in long room 601, so that allowing small molecule to diffuse into and leave can be in the hollow fibre in room 601 Dimension.

The pressure/temperature sensor 624 being arranged in second fluid circulating path 604 allows to enter cell in culture medium raw The pressure and temperature of culture medium is measured before the space EC of long room 601.Sensor 626 allows in second fluid circulating path 604 In culture medium its pressure and/or temperature are measured after leaving cell growth chamber 601.It, can be in the operation phase about the circuit EC Between from sample port 630 or sample coil obtain media samples.

After leaving the EC outlet port 601D of cell growth chamber 601, the fluid in second fluid circulating path 604 is logical It crosses EC circulating pump 628 and reaches oxygenator or gas transport module 632.According to embodiment, EC circulating pump 628 can also be along opposite Direction pumps fluid.Second fluid flows path 622 can be via oxygenator or the ingress port 632A of gas transport module 632 It is connected with outlet port 632B with oxygenator or 632 fluid of gas transport module.In operation, broth is via arrival end Mouth 632A flows into oxygenator or gas transport module 632, and leaves oxygenator or gas transport module via outlet port 632B 632.For example, oxygenator or gas transport module 632 will go degasification in culture medium that oxygen is added in CES 600 and therefrom Bubble.In various embodiments, the culture medium in second fluid circulating path 604 can be with entrance oxygenator or gas transport mould The gas of block 632 balances each other.Oxygenator or gas transport module 632 can be any appropriate ruler for oxygen conjunction or gas transport Very little device.Air or gas flows into oxygenator or gas transport module 632 by filter 638, and is flowed by filter 640 Oxygenator or charge delivery mechanism 632 out.Filter 638 and 640 reduces or prevents oxygenator or gas transport module 632 and phase Close the pollution of culture medium.Oxygenator or gas can be passed through from the air or gas that CES 600 is purified during the part of perfusion event Body shift module 632 is discharged into atmosphere.

In the configuration described for CES 600, in first fluid circulating path 602 and second fluid circulating path 604 Broth cell growth chamber 601 is flowed through with the same direction (and stream configuration).According to embodiment, CES 600 can be with It is configured to flow with the structure of convection current.

According at least one embodiment, culture medium including cell (deriving from such as cell container, such as bag) can be with It is attached to attachment point 662, and the broth from culture medium source can be attached to attachment point 646.It can be via first Cell and culture medium are introduced first fluid circulating path 602 by fluid flow path 606.Attachment point 662 can via valve 664 with 606 fluid of first fluid flow path is connected, and attachment point 646 can be flowed via valve 650 and first fluid flow path 606 Body is connected.Reagent source can be fluidly connected to a little 644 and by valve 648 and fluid inlet path 642, or pass through valve 648 It is connected with 672 with 674 fluid of second fluid ingress path.

Air removes room (ARC) 656 can be connected with 602 fluid of first circulation path.Air removes room 656 One or more ultrasonic sensors, ultrasonic sensor include upper sensor and lower sensor to detect air, fluid Lack and/or air/fluid interface is (for example, gas/fluid circle at certain measurement positions in air removal room 656 Face).For example, can be near the bottom that air removes room 656 and/or near top using ultrasonic sensor detects these Air, fluid and/or air/fluid interface at position.Without departing from the spirit and scope of the present invention, embodiment party Case provides the use of many other types of sensor.For example, optical sensing can be used with embodiment according to the present invention Device.It can be discharged into from the air or gas that CES 600 is purified via pipeline 658 during the part of perfusion event or other schemes Atmosphere outside air valve 660, pipeline 658 can remove 656 fluid of room with air and be connected.

EC culture medium source can be attached to EC culture medium attachment point 668, and wash source of solvent can be attached to washing it is molten EC culture medium and/or washing solution are added to first or second fluid flow path by liquid attachment point 666.Attachment point 666 can To be connected with 670 fluid of valve, valve 670 can be via valve 672 and first fluid ingress path 642 and first fluid circulating path 602 fluids are connected.Alternatively, attachment point 666 can be via second fluid ingress path by opening valve 670 and closing valve 672 674 and second fluid flowing path 684 be connected with 604 fluid of second fluid circulating path.Similarly, tie point 668 can be with 676 fluid of valve is connected, and valve 676 can be flowed via first fluid ingress path 642 and valve 672 and first fluid circulating path 602 Body is connected.Alternatively, fluid container 668 can be flowed with second fluid ingress path 674 by opening valve 676 and closing distributing valve 672 Body is connected.

In the circuit IC, fluid can be promoted initially by IC inlet pump 654.In the circuit EC, fluid can be initially by EC Inlet pump 678 promotes.The air monitor 580 of such as ultrasonic sensor can also be associated with EC ingress path 684.

In at least one embodiment, the first and second fluid circulation paths 602 and 604 are connected to waste line 688. When valve 690 is opened, IC culture medium can flow through waste line 688 and flow to litter bag or outlet pocket 686.Equally, work as valve When 692 opening, EC culture medium can flow to litter bag or outlet pocket 686.

After cell is grown in cell growth chamber 601, path 697 can be harvested by cell and harvest cell.Herein, It, in the case where valve 698 is opened, can be pumped to thin by the way that IC culture medium containing cell is harvested path 697 by cell Born of the same parents' harvest bag 699 harvests the cell from cell growth chamber 601.

The various parts of CES 600 may include or be contained in machine or shell (such as cell amplification machine 202 (Fig. 2 and 3)), wherein for example maintaining the machine of cell and culture medium at a predetermined temperature.It, can be with it is further to be noted that in embodiments Combine the component of CES 600 and CES 500 (Fig. 5).In other embodiments, CES may include than shown in Figures 5 and 6 The less or additional component of component, and still be within the scope of the present invention.System can be expanded in conjunction with the cell of feature of the invention The example of system isCell expansion system is manufactured by the Terumo BCT company of state of Colorado Lakewood.

In United States Patent (USP) No.8,309,347 (" cell expansion system and its application method ", publications on November 13rd, 2012) In provide cell expansion system example and further describe, be incorporated herein in its entirety by reference as its introduction and For all purposes.

Although it have been described that the various example embodiments and relative method of cell expansion system, according to this hair Bright embodiment, Fig. 7 show the component (for example, EV or viral vectors etc.) for generating, purifying and/or collecting release The exemplary operation step 700 of process, can be with cell expansion system (such as CES 500 (Fig. 5) or (figure of CES 600 6) it) is used together.Starting starts operation 702, and process 700 proceeds to the inoculating cell 704 in bioreactor.At one In embodiment, in the 0th day inoculating cell.In one embodiment, it is inoculated with MSC.As understood by those skilled in the art, Any cell type of cellular products (such as EV or viral vectors etc.) needed for discharging can be used.

Next, according to an embodiment, 706 cells can be expanded with culture medium until converging.In another implementation In scheme, cell can expand the number until the multiplication of cell needed for occurring, for example, the multiplication of cell, cell multiplication twice, Cell multiplication, the multiplication of four cells, five cells multiplications, six times or more time cell multiplications etc. three times.In another embodiment party In case, cell can expand specific a period of time.For example, cell can expand for about 24 (24) hours to about 48 (48) time of hour.In another embodiment, cell can expand for about 48 (48) hours to about 72 (72) The time of hour.In another embodiment, cell can expand for about 24 (24) hours to about 72 (72) hours Time.In embodiments, this amplification for example occurred by the 3rd day on day 1.According to another embodiment, cell can To expand the time for being less than about for 24 (24) hours.In another embodiment, cell can be expanded greater than 72 (72) time of hour.For example, in one embodiment, cell can expand about seven (7) to about before collecting excretion body Eight (8) days.Any a period of time can be used with embodiment according to the present invention.

For example, complete medium amplifying cells 706 can be used.In one embodiment, the culture medium includes to contain blood Clear culture medium (such as α-MEM) and serum.In one embodiment, the serum of animal origin can be used.At another In embodiment, source of people serum can be used.In another embodiment, synthesis serum can be used.In another implementation In scheme, another type of serum can be used.The example of serum-containing media includes α-MEM+GlutaMAX+10% tire ox Serum (FBS).In a further embodiment, serum free medium can be used.Those skilled in the art can be used Any kind of culture medium known.

Fig. 7 is returned to, process 700 next moves on such as optional step 708, for flushing out the first culture medium (such as Any culture medium containing serum).In one embodiment, the culture medium containing serum with the second culture medium (such as basis train Support base) substitution.The example of basal medium includes α-MEM+GlutaMAX.It can be used well known by persons skilled in the art any The culture medium of type.In order to from the release ingredient of isolated or purified in the cell of amplification (in serum or other protein sources Existing any ingredient control), according to embodiment, cleaning procedure removes any serum (such as haemocyanin).In a reality It applies in scheme, cleaning procedure occurs on day 3.In one embodiment, when for example, not using the serum of animal origin, only Using source of people serum, using serum free medium, and/or for example, it is removed without additional protein source as other In the case of, step 708 can be optionally.

Next, process 700 continue component that release is collected in circuit (such as the circuit IC) or releaser 710 (such as EV or viral vectors etc.).In one embodiment, by closing the outlet IC (such as closing IC outlet valve) for the component of release Or substance (such as EV or viral vectors etc.) is concentrated in the circuit IC, to carry out this collection.This concentration can occur one Time as defined in section.In one embodiment, such period may include that the component that will be discharged is concentrated greatly in the circuit IC About 48 (48) hours.In another embodiment, such period may include the component concentration about two that will be discharged 14 (24) hours to about 48 (48) hours.In another embodiment, such period may include that will discharge Component be concentrated about 24 (24) hours to about 72 (72) hours.In another embodiment, when such Between section may include will discharge component concentration about 48 (48) hours to about 72 (72) hours.In an embodiment party In case, such period may include that the component concentration that will be discharged was greater than about for 72 (72) hours.In one embodiment, For example, this collection occurs on day 3 by the 5th day.Also in another embodiment, such period may include that will discharge Component be concentrated less than about 24 (24) hours.Any time section can be used with embodiment according to the present invention.In this way Collection during, cell can supplement nonprotein culture medium, wherein this culture medium can be added and spread from the side EC Pass through semi-permeable membrane.In another embodiment, for example, culture medium can be added from the side IC, wherein cell can be supplemented and is free of The culture medium of protein.

After collecting the component of release in the circuit IC (or EC), process 700 continues the component 712 of harvest release.One In a embodiment, this harvest for example occurred at the 5th day.In one embodiment, component (such as EV or the virus of release Carrier etc.) form that can the suspend circulation loop out of capillary in bioreactor is transferred to harvest bag or harvest container In.In one embodiment, nonprotein culture medium can be used for such harvest plan.

Next, process 700 optionally proceeds to further process step 714, wherein for example can handle harvest The component of release is for measuring.In another embodiment, this further processing 714 may include from harvest bag The further concentration of the releaser of the culture medium of middle collection.In another embodiment, such as in the feelings using suspension cell Under condition, such to be further processed 714 may include separating releaser with the other components (such as cell) in bag, to receive Obtain releaser.In one embodiment, it is this be further processed 714 may include release component it is further separation with/ Or characterization.From optionally step 714 is further processed, process 700 can terminate at end operation 716.Alternatively, process 700 It can be directly to end operation 716 from harvest step 712, and not need optionally to be further processed step 714 In the case of terminate.

Fig. 8 is described next, will be arranged in conjunction with example with culture medium introduction.However, embodiment given here is not It is limited to the embodiment, but embodiment can be modified to meet other requirements or system design or configuration.Starting starts to operate 802, and process 800 continues for disposable tubing group 804 to be loaded into cell expansion system.

Next, system can be poured 806.In one embodiment, for example, user or operator can pass through choosing It selects and for example comes to provide the instruction for perfusion to system for the task of perfusion.In one embodiment, this for being perfused Task can be the task of pre-programmed.System 500 (Fig. 5) or 600 (Fig. 6) can be for example with phosphate buffered saline (PBS) (PBS) Perfusion.In order to which bioreactor 501,601 is perfused, bag (546) can be attached (for example, being connected to tie point 646) and arrive system 500,600.When the number in reference attached drawing, for example, such as " number, number " (for example, 500,600), such term can To indicate " number and/or number " (for example, 500 and/or 600).Valve 550,650 can be opened.It may then pass through IC entrance Pump 554,654 starts to be pumped into PBS in first fluid circulating path 502,602, and PBS is pumped into first fluid the circulation path In diameter 502,602.It, can when PBS enters through entrance 501A, 601A bioreactor 601 and flows out from outlet 501B, 601B To open valve 614.According to an embodiment, once bioreactor 501,601 and/or first fluid circulating path 502, 602 wherein have the culture medium that the removal of room 556,656 air is removed by air, and bioreactor 501,601 is just poured.

In order to which bioreactor 501,601 is further perfused, bag 586 can be attached (for example, being connected to tie point 668) To system 500,600.Valve 576,676 can be opened.It may then pass through IC inlet pump 554,654 to start for PBS to be pumped into In one fluid circulation paths 502,602, and PBS is pumped into first fluid circulating path 504,604.When PBS passes through entrance When 501C, 601C enter bioreactor 601 and flow out from outlet 501D, the 601D in the circuit EC, valve 692 can be closed.According to One embodiment passes through once bioreactor 501,601 and/or second fluid circulating path 504,604 have wherein Air removes the culture medium that room 580,680 removes air, and bioreactor 501,601 is just poured.

Then, process 800 is carried out to coating bioreactor 808, and wherein bioreactor 501,601 can be coated with and apply Layer agent (such as 5mg fibronectin (FN)).For example, in embodiments, reagent bag 544 can be loaded (for example, in tie point On 644) into IC ring 502,602, until reagent container 544 is sky.Room 556,656 can be removed from air to draw reagent 544 Into the circuit (chased) IC 502,602, may then pass through operation circulation pump 512,612 and/or inlet pump 554,654 makes to try Agent 544 recycles in the circuit IC 502,602.Can be used any coated agent well known by persons skilled in the art (such as FN or Cool insoluble protein).

The system 500,600 for introducing bioreactor can be as follows with the example for coating the solution of bioreactor:

Table 1

The coating of bioreactor can carry out in three phases.System for introducing the first stage of above-mentioned solution 500, the example of 600 setting can be as follows:

Table 2

For coat from air remove room 556,656 in capture reagent bioreactor second stage system 500, The example of 600 setting can be as follows:

Table 3

For being coated in the system 500,600 for recycling the phase III of bioreactor of reagent in the circuit IC 502,602 Setting example, can be as follows:

Table 4

Once being coated with bioreactor, so that it may execute IC/EC in step 810 and rinse task, wherein can replace Fluid on IC circulation loop 502,602 and on EC circulation loop 504,604.Replace volume can by the IC volume of exchange and The quantity of EC volume determines.The solution example that CES 500,600 is introduced during IC/EC flushing task is as follows:

Table 5

The example that the IC/EC of system 500,600 rinses the setting of task can be as follows:

Table 6

Next, can be held for the appropriate or required gas concentration being maintained across in bioreactor film on fiber Row conditioned medium task 812 is to allow culture medium to supply before by loading cells into bioreactor with provided gas Balance should be reached.For example, providing culture medium by using high EC cycle rate and by gas transport module or oxygenator 532,632 Rapid contact between the gas supply of offer.Then, system 500,600 may remain in appropriate or required state, until Such as user or operator prepare loading cells into bioreactor 501,601.In one embodiment, CES 500, 600 can for example be adjusted with complete medium.Complete medium can be any culture medium source for cell growth.One In a embodiment, complete medium may include such as α-MEM and fetal calf serum (FBS).Those skilled in the art can be used Known any kind of culture medium.

Conditioned medium task 812 can be the process of two steps, in the first step, system 500,600 by using High EC cycle rate provides the Rapid contact between culture medium and gas supply.In the second step, system 500,600 will be biological Reactor 501,601 maintains state appropriate, until operator is ready to load cell.In 812 phase of conditioned medium task Between introduce the example of solution of CES 500,600 can be as follows:

Table 7

The example of the setting of the first step of conditioned medium task 812 can be as follows:

Table 8

The example of the setting of the second step of conditioned medium task 812 can be as follows:

Table 9

For example cell 814 is loaded into from cell entry bag 562 (at tie point 662) next, process 800 proceeds to In bioreactor 501,601.Loading cell can be three-step process.Firstly, for example cell can from cell entry bag 562 with Uniform suspension 814 is loaded into bioreactor 501,601 (at tie point 662), until bag 562 is sky.Another In a embodiment, cell can be loaded for example, by another type of load module (such as passing through target center load module) 814.Any kind of load module can be used according to embodiment.Secondly, then cell can be removed room from air 556,656 bioreactor 501,601 is captured.In the configuration using bigger trapping volume, cell can be spread and court It is mobile to the outlet IC 590,690.In third step, for example, the IC circulation by no IC entrance (such as passes through IC circulating pump 514,614), can promote cell is distributed through film.

Introducing CES 500,600 can be as follows with the example for loading the solution of cell 814:

Table 10

As described above, loading cell 814 can occur in three phases.System 500,600 for the first stage The example of setting can be as follows:

Table 11

The example of the setting of system 500,600 for second stage can be as follows:

Table 12

The example of the setting of system 500,600 for the phase III can be as follows:

Table 13

Further, for example, can permit cell (for example, attached cell) attachment 816 to doughnut.In an embodiment party In case, when allowing cell to adhere to 816, attached cell can be attached on bioreactor film, while allow to be recycled back in EC It is flowed on road 504,604, the flow set in 514,614 to the circuit IC 502,602 of pump is zero.The mistake of film 816 is attached in cell The example that the solution of CES 500,600 is introduced in journey can be as follows:

Table 14

Example for attachment to the setting of the film 816 in system 500,600 can be as follows:

Table 15

Cell can be supported in step 818, wherein IC circulation can be added continuously to flow velocity (for example, low flow velocity) Circuit 502,602 and/or EC circulation loop 504,604.The fluid that outlet setting allows to be added to system 500,600 removes.? Support the example for introducing the solution of system 500,600 during step 818 can be as follows:

Table 16

The example of the setting of keeping step 818 in system 500,600 can be as follows:

Table 17

Next, process 800 may proceed to optional step 820 to flush out any culture medium containing serum and use base Basal culture medium or the replacement of nonprotein culture medium.If previous processing is loaded carefully using nonprotein culture medium Born of the same parents, keeping cell etc., then may not be needed step 820.However, if using the protein containing serum, such as can be thin Born of the same parents reach converge after, reaching the defined period, or after the number for reaching certain required cell multiplication, at expected point From and/or collect release cellular products (for example, EV or viral vectors etc.) after start cleaning procedure 820.

For example, it may be desirable in the multiplication of least twice cell, three times cell multiplication, four cell multiplications, five cells times Increase, after six times or more cell multiplications etc., starts the purifying and/or collection of the cellular products of release.Can be used one or The culture medium that more processes are come in purification system 500,600 is to collect the EV product of release.Such purifying procedure may include 5X IC EC rinses 820, negative ultrafiltration and rinses 822, IC EC flushing 824 and/or another type of cleaning procedure.Firstly, 5X IC It may include replacing 502,602 and EC of IC circulation loop circulation loop 504, the fluid in 604 the two that EC, which rinses 820, wherein can To replace volume by IC volume and the quantity of EC capacity exchange come specified.

The example that the solution of system 500,600 is introduced during 5x IC/EC rinses task 820 can be as follows:

Table 18

The example that the 5x IC/EC of system 500,600 rinses the setting of task 820 can be as follows:

Table 19

Further, it may include using negative ultrafiltration washing IC circulation loop 502,602 to help that optional negative ultrafiltration, which rinses 822, Help promotion that may have been deposited on any component of the side IC of fiber.The solution of CES 500,600 is introduced during negative ultrafiltration 822 Example can be as follows:

Table 20

The example of the setting of the negative ultrafiltration 822 of system 500,600 can be as follows:

Table 21

Further, optional IC EC rinse 824 can be used for replacing 502,602 and EC of IC circulation loop circulation loop 504, Fluid on 604, wherein replacement volume can be specified replacement volume by the quantity of IC volume and EC volume exchange.In this flushing In program, culture medium used be can be, such as matrix culture medium or nonprotein culture medium.It is rushed in optional IC EC The example that the solution of system 500,600 is introduced during washing 824 can be as follows:

Table 22

The example that the IC EC of system 500,600 rinses 824 setting can be as follows:

Table 23

These above-mentioned tasks can be client or customized task.In other configurations, such task be can be Pre-programmed or default task.In other embodiments, such task can be by, such as user or operator hold manually Row.

In embodiments, for example, (for example, being rinsed before 5x flushing, in 5x at the difference during cleaning procedure After 820, negative ultrafiltration rinse 822 after, basal medium exchange 824 after) other configurations in, system 500,600 In gross protein can be measured in the side IC (or the side EC), for proving the removal of serum or haemocyanin (for example, going completely Except).Further, for example, in the serum for not using animal origin, source of people serum is used only, using serum free medium, and/ Or for example, being removed without other such additional protein sources in the case where, step 820,822 and/or 824 can be Optionally.

According to an embodiment, the effect of above method, can be indicated by the chart 1100 in Figure 11.Figure 110 0 is indicated Implement the possible outcome of protein measurement in the system (such as system 500,600) of above procedure.As shown, system 500, The amount of protein in 600 can be indicated by line 1104.Egg before above procedure 820,822,824, in system 500,600 The amount of white matter may be at peak value 1108 (for example, more than 7000 μ g/mL).During 5x rinses 820, the amount of protein can be steady Drop is fixed, as shown in the part 1112 of line 1104.In fact, protein concentration can be substantially at the end of 5x rinses 820 0 μ g/mL, as shown in the part 1116 of line 1104.In this case, it is possible to not need for example, 822 or basis are rinsed in negative ultrafiltration Culture medium exchange 824.However, according to an embodiment, program 822 and 824 can effectively further extraction system 500, Protein in 600.

It, can be by the culture medium of addition to biological anti-after washing away any culture medium containing serum in bioreactor Answer the side EC 504,604 of device 502,602 and diffuse through semi-permeable membrane, by bioreactor cell and nonprotein training Feeding base supports 826 defined a period of times (for example, about 48 (48) hours) together.EC entrance 668 can supply EC culture medium (such as nonprotein culture medium) is used for cells with nutrient, while the cellular products concentration for allowing to discharge.This In configuration, may there is no IC entrance.Instead, the semipermeable hollow fibers of bioreactor 502,602 allow basic nutrition object (for example, glucose) reaches cell by continuous pouring, and can actively remove metabolic waste (such as lactic acid) and can be with By diffusing out system 500,600 (EC exit opening 582,692).This keeping can occur one section as defined in time (example Such as about 48 (48) hours).In another case, extremely using about 24 (24) hour of nonprotein culture medium About 72 (72) hours are to supplement cell.In another embodiment, with nonprotein culture medium (such as second training Support base) this keeping is carried out, for about 48 (48) hours to about 72 (72) hours to supplement cell.In another reality It applies in scheme, carrying out this keeping with the second culture medium (such as nonprotein culture medium), to can last about 24 (24) small Up to about 48 (48) hours.In another embodiment, this keeping about 24 is carried out with the second culture medium (24) hour to about 48 (48) hours.Also in another embodiment, this is carried out with nonprotein culture medium Less than about 24 (24) hours time that kind is supported.According to embodiment, the second culture medium can be used and support any time Section.In another arrangement, IC entrance can provide IC culture medium (such as nonprotein culture medium), for mentioning to cell For nutrition.According to an embodiment of the invention, any time section can occur for this keeping.This keeping 826 of cell can wrap It includes through closure IC outlet valve 590,690 and closes IC entrance.The molten of system 500,600 is introduced during the keeping 826 of cell The example of liquid can be as follows:

Table 24

The example of the setting of the keeping of cell 826 with system 500,600 can be as follows:

Table 25

In another embodiment, occur for example, closing the outlet IC and allowing to collect the cellular products discharged in step 828 In.The cellular products that this closing (by closing valve 590,598 or 690,698) of the outlet IC allows cell to discharge are biological anti- It answers and 828 is collected or be concentrated in device.Allowed through the side EC 504,604 actively by keeping the outlet EC to open (valve 582,692) ultrafiltration Remove waste.Further, semi-permeable membrane allow must nutrients cell reached by continuous pouring.As described in table 24 and 25, For example, according to an embodiment, it can be by optionally thin to support in the side IC addition culture medium (for example, without protein) Born of the same parents 826.In one embodiment, culture medium bag 546 may be coupled to tie point 646.From bag 546 culture medium (for example, Without protein) IC suction line 506,606 can be admitted to by valve 550,650.Culture medium can from suction line 506, 606 enter the cell in the part IC of the circuit IC 502,602 for supporting bioreactor 501,601.

Additionally or alternatively, step 826 and/or 828 may also include outlet or litter bag 586,686 optional alternative 827,829.In one embodiment, outlet or this replacement of litter bag 586,686, allowing to monitor or test will be supervised The content of glucose/lactic acid production bag is surveyed, whether passes through film (because the outlet IC is closed with the cellular products of any release of determination Close) etc..As described above, cellular products (such as EV or viral vectors) generated by cell etc. may cannot diffuse through greatly very much Film (for example, its molecular weight is too big).In the case where IC is exported and closed, the cellular products (such as EV) of release can be in release Amplification (or the collection phase limited) period in the case that the concentration of cellular products (such as EV) is continuously increased is maintained at biological anti- Answer the side IC of device 501,601.The keeping 826 of cell can occur simultaneously with the IC closing exported.In another embodiment, Collection step 828 occurs after for example closing the outlet IC.Also in another embodiment, step 826 is for example collecting step Rapid 828 close generation before the outlet IC.In other embodiments, test/monitoring feelings are not needed after the outlet IC is closed Under condition, it is changed without outlet pocket 586,686.In yet another embodiment, outlet or waste canister or bag can be used for collecting and receiving Obtain releaser.Although the outlet IC is considered closing in process 800, it should be noted that for example, in other embodiments, in the side EC In the case where cell growth can occur, the outlet EC can be closing.

According to an embodiment, start to collect concentration in circuit (such as circuit IC 502,602) or collect when determining Cellular products 828 when (such as after about 48 (48) hours, or for example, another a period of time), the valve for harvest 598,830 are open 698, and can be harvested from the side IC of bioreactor 501,601 502,602 cellular products 830 to Harvest container 599,699.In one embodiment, the IC valve 590,690 that IC exports 586,686 is opened to allow to harvest.? In another embodiment, harvest valve 598,698 is opened to allow to harvest harvest bag 599,699 or harvest container.In a reality It applies in scheme, this harvest occurs in the last of whole process.In another embodiment, this to harvest at defined intervals Occur.In one embodiment, cellular products (such as EV or viral vectors etc.) can be with suspended pattern from bioreactor 501, circulation loop 502,602 is transferred in harvest bag 599,699 or harvest container in the capillary in 601.Implement at one In scheme, nonprotein culture medium can be used for such harvest task.During collecting concentration or the cellular products collected 828 The example of the solution of introducing system 500,600 can be as follows:

Table 26

The example of the setting of the collection of concentration and collection cellular products with system 500,600 can be as follows:

Table 27

After harvesting releaser 830, in the case where not needing to be further processed 832 or harvest cell 836, process 800 can directly carry out from harvest cellular products 830 to terminate at end operation 838.

Alternatively, process 800 is optionally carried out to allow to be further processed 832 from cutting 830.In an embodiment In, it is such that be further processed 832 may include the processing for measurement.In another embodiment, such to be further processed 832 It may include the further concentration of the cellular products for the culture medium or fluid collected in harvest bag.In another embodiment In, can be with amplifying suspended cell and in the case where harvest release product, such to be further processed 832 may include producing cell Object is separated with the other components (such as cell) in bag.It is in one embodiment, such that be further processed 832 may include releasing The further separation for the cellular products put and/or characterization.From optionally step 832 is further processed, retain not needing harvest In the case where cell (such as any attached cell) in the bioreactor, process 800 can be whole at end operation 838 Only.

On the other hand, retain cell in the bioreactor (for example, any attached cell or attachment if necessary to harvest Cell), then process 800 carry out release cell 834.Alternatively, not needing to be further processed 832 and need from biological respinse In the case that device discharges cell 834, process 800 can directly carry out discharging cell 834 from harvest cellular products 830.For example, attached Cell can discharge and 834 and be suspended in the circuit IC from the film of bioreactor.In embodiments, prepare addition reagent IC/EC is carried out to discharge cell rinses task as a part of operation 834.For example, prepare addition trypsase or its When his chemoreleaser is to discharge any attached cell, IC/EC culture medium can be replaced with phosphate buffered saline (PBS) (PBS) to go Isolating protein, calcium (Ca²⁺) and magnesium (Mg²⁺).Can by load reagents into system until reagent bag be sky.Reagent can be caught Receive in the circuit IC, and can in the circuit IC mix reagent.After discharging any attached cell, harvest operation 836 will hang Floating cell is transferred in harvest bag or container from IC circulation loop (e.g., including stay any cell in the bioreactor). Finally, then process 800 terminates at end operation 838.

Next, Fig. 9 shows embodiment according to the present invention, for generating, separating and/or collecting releaser The exemplary operation step 900 of the process of (such as EV or viral vectors etc.), can be used together (all with cell expansion system Such as CES 500 (Fig. 5) or CES 600 (Fig. 6)).Starting starts operation 902, and process 900 is carried out disposable tubing group 904 are loaded into cell expansion system.Next, system can be perfused 906.In one embodiment, for example, user or behaviour Author can provide the instruction for perfusion to system by selecting the task for perfusion.In one embodiment, this Kind can be the task of pre-programmed for the task of perfusion.Then, process 900 carries out coating bioreactor 908, wherein biology Reactor can be coated with coating agent.For example, in embodiments, can by load reagents into the circuit IC until reagent container For sky.Reagent can be captured in IC loop from air removal room, then can recycle reagent in IC loop.It can be used For example any coated agent (such as fibronectin or cool insoluble protein) well known by persons skilled in the art.Once bioreactor quilt Coating, so that it may execute IC/EC and rinse task 910, wherein the fluid on IC circulation loop and on EC circulation loop can be replaced. According to an embodiment, replacement volume can be determined by the quantity of the IC volume and EC volume that exchange.

Next, can be held for the appropriate or required gas concentration being maintained across in bioreactor film on fiber Row conditioned medium task 912 is to allow culture medium to supply before by loading cells into bioreactor with provided gas Balance should be reached.For example, provided by using high EC cycle rate offer culture medium and by gas transport module or oxygenator Rapid contact between gas supply.Then system can be maintained to appropriate or required state, until such as user or behaviour Author prepares loading cells into bioreactor.In one embodiment, for example, system can use such as culture completely Base is adjusted.Complete medium can be any culture medium source for cell growth.In one embodiment, training completely Feeding base may include such as α-MEM and fetal calf serum (FBS).Any kind of culture well known by persons skilled in the art can be used Base.

Next, process 900 continues for cell to be downloaded to 914 in bioreactor from such as cell entry is packed.At one In embodiment, cell can be downloaded in bioreactor from cell entry is packed until bag is sky.Then can by cell from Air removal room captures bioreactor.In the embodiment using biggish trapping volume, cell can stretch and court It exports and moves to IC.Cell distribution is promoted to pass through film for example, by the IC circulation (such as passing through IC circulating pump) of no IC entrance. It is possible to further allow cell (such as attached cell) attachment 916 to doughnut and support 918.In an embodiment In, in the case where allowing cell to adhere to 916, attached cell can be attached on bioreactor film while allow to flow in EC Pump discharge on circulation loop to the circuit IC is set as zero.Cell can grow/expand 920.In one embodiment, cell Three (3) can be expanded to four (4) days.In another embodiment, cell can expand a period of time to realize specific institute The cell of number is needed to double.In another embodiment, cell, which can expand, is converged for a period of time with reaching.Implement at one In scheme, cell can expand about seven (7) to about eight (8) days before collecting EV (such as excretion body).It can be according to the present invention Embodiment use any time section.

Next, process 900 proceeds to inquiry 922, where it is determined whether need to collect during cell growth/amplification by Cell is discharged into the cellular products (such as EV or viral vectors etc.) in conditioned medium.For example, it may be desirable in specific or limit Determine the cell times of number (such as double twice, double three times, four multiplications, five multiplications, six times or more cell multiplications) Releaser is initially separated or purified after increasing for collecting.For example, in one embodiment, it may be desirable in least twice cell Releaser is initially separated and/or collected after multiplication.In another embodiment, for example, it may be desirable in minimum five cells Start to purify and/or collect releaser after multiplication.In one embodiment, before being initially separated and/or collecting releaser, The cell multiplication for limiting number can not be set.As understood by those skilled in the art, the cell of any number can be used Multiplication, the period limited and/or other indicants.

Back to inquiry 922, if necessary to separate and/or collect releaser, then "Yes" is branched off into optionally by process 900 It rinses and replacement step 924.In one embodiment, the culture medium containing serum can be flushed out, wherein this culture Base can be replaced with basal medium, for example, 924.In one embodiment, this cleaning procedure may comprise steps of In it is one or more: (1) 5X IC EC rinse；(2) negative ultrafiltration flushing is carried out with phosphate buffered saline (PBS) (PBS), with to the greatest extent May serum deprivation (if any) be removed from bioreactor morely；And/or (3) rinse 2.5X IC EC with basal medium To supplement any metabolin lost during PBS is rinsed.In one embodiment, all (1)-listed above (3) are executed Step.In another embodiment, one of (1)-listed above (3) step is only executed.In another embodiment, into Two steps in row (1)-listed above (3) step.In addition, any sequence of steps can be used according to embodiment.? In further embodiment, when for example, being trained without using the serum of animal origin, using only source of people serum, using serum-free Support base, and/or for example, being removed without additional protein source as other in the case where, in step 924, there is no punchings It washes, and such step 924 can be optionally.

After flushing out any culture medium 924 containing serum in bioreactor, closing IC outlet valve can be passed through 926 are exported to close IC, to allow cell releaser concentration to increase in the bioreactor.One as such step 926 Part can optionally replace outlet or litter bag 928.Such outlet or the replacement of litter bag allow the interior of sack to be executed Tolerant monitoring or test determine whether any releaser passes through film (because the outlet IC is closed) to monitor glucose/lactic acid production Deng.As described above, releaser (such as EV or viral vectors) generated by cell etc. may cannot diffuse through film (example greatly very much Such as, molecular weight is too big).According to embodiment, in the case where IC is exported and closed, releaser (such as EV) can be in releaser Amplification (or the collection phase limited) period in the case that the concentration of (such as EV or viral vectors etc.) continues to increase is maintained at raw The side IC of object reactor.In one embodiment, the closing that step 928 is exported with IC occurs simultaneously.In another embodiment party In case, step 928 occurs after closing the outlet IC.Also in another embodiment, step 928 occurs closing the outlet IC Before.In other embodiments, after the outlet IC is closed, test/monitoring is not being needed or other are specific using outlet pocket In the case where, outlet pocket can be changed without.Although the outlet IC is closes in process 900, it should be noted that for example, in other realities It applies in scheme, in the case where cell growth can occur for the side EC, the outlet EC is to close.

Next, process 900 is carried out to support cell 930 and collect releaser 932, wherein can will be nonprotein Culture medium is added to the side EC of bioreactor and can diffuse through semi-permeable membrane to the side IC.In such an implementation, EC Entrance may include EC culture medium (such as nonprotein culture medium) and be used for cells with nutrient, while allowing to discharge Cellular products concentration.In such an implementation, for example, it may be possible to there is no IC entrance.Instead, in the semi permeability of bioreactor Hollow fiber allows necessary nutrients (for example, glucose) to reach cell by continuous pouring, and it is useless actively to remove metabolism Object (such as lactic acid) and can be by diffusing out system (EC port opening).In one embodiment, can by Culture medium is added on the side IC supports cell.In one embodiment, using about 48 (48) hour of basal medium to mend Fill cell.In another embodiment, using about 24 (24) hour of basal medium to about 72 (72) hours with Supplement cell.In embodiments, in IC loop (IC outlet close 926) releaser (such as EV or viral vectors etc.) collection Or the period that concentration can limit, for example, about 48 (48) hours, or in other embodiments, such as About 24 (24) hours to about 72 (72) hours.In other embodiments, this to be supported with basal medium It can carry out greater than about 72 (72) hours that (such as, about 72 (72) hours are to about with release particle is collected 96 (96) hours).In another embodiment, about seven can be carried out with the second medium feed and collection release particle 12 (72) hours to about 120 (120) hours.In other embodiments, this charging and collection can be less than The period of 24 (24) hours.

According to an embodiment, when determine start releaser that harvest is collected or concentration when (such as about 40 After eight (48) hours, such as after another period of time), the valve 934 for harvest can be opened, and can incite somebody to action Releaser 936 is harvested from the side IC of bioreactor into harvest container.In one embodiment, it can open to go out for IC The IC valve of mouth is to allow to harvest.In another embodiment, harvest valve can be opened to allow to harvest to harvest bag or harvest Container.In one embodiment, this harvest can occur at the end of whole process.In another embodiment, this Kind harvest can occur at defined intervals.In one embodiment, releaser (such as EV or viral vectors etc.) can hang Floating form is transferred in harvest bag or harvest container from circulation loop in the capillary in bioreactor.In an embodiment party In case, nonprotein culture medium can be used for such harvest task.

After harvesting releaser 936, process 900 can be carried out optionally to allow to be further processed 944.In a reality It applies in scheme, this further processing 944 may include the processing for measurement.In another embodiment, it is this into The processing 944 of one step may include the further concentration of the releaser from the culture medium collected in harvest bag.In another reality It applies in scheme, in the case where suspension cell growth and harvest releaser, such to be further processed 944 may include by releaser It is separated with the other components (such as cell) in bag.It is in one embodiment, such that be further processed 944 may include releaser It is further separation and/or characterization.From optionally step 944 is further processed, bioreactor is retained in not needing harvest In cell (such as any attached cell) in the case where, process 900 can terminate at end operation 946.In another reality It applies in scheme, in the case where not needing to be further processed 944 or harvest cell 940, process 900 can be directly from cutting 936 carry out to terminate at end operation 946.

Back to step 944, if necessary to harvest for example, the cell of reservation in the bioreactor is (for example, any adherency Or the cell of attachment), then process 900 proceeds to optional release cell step 938.Alternatively, not needing to be further processed 944, and need from bioreactor discharge cell 938 in the case where, process 900 can directly proceed to from cutting 936 Optional release cell step 938.For example, attached cell can discharge 938 from the film of bioreactor and be suspended in the circuit IC. In embodiments, prepare addition reagent and carry out IC/EC flushing task to discharge cell as a part of operation 938.Example Such as, when preparing addition trypsase or other chemoreleasers to discharge any attached cell, IC/EC culture medium can use phosphorus Hydrochlorate buffered saline (PBS) is replaced to remove isolating protein, calcium (Ca²⁺) and magnesium (Mg²⁺).It can be straight into system by load reagents It is sky to reagent bag.Reagent can be captured in the circuit IC, and can in the circuit IC mix reagent.Discharging any patch After parietal cell, harvest operation 940 by suspension cell from IC circulation loop (e.g., including stay in the bioreactor any thin Born of the same parents) it is transferred in harvest bag or container.Finally, disposable setting optionally can be used as process from cell expansion system unloading The 942 of 900 a part, then process 900 terminates at end operation 946.

The process shown in Fig. 7,8 and 9, embodiment according to the present invention, discribed operating procedure be for The purpose of explanation and provide, and can be rearranged, be combined into other steps, be used parallel with other steps.? In the case where not departing from the spirit and scope of the present invention, less or additional step can be used in embodiments.In addition, In some embodiments, such as by executing the processor of pre-programmed mission stored in memory, step can be performed automatically Suddenly (and any sub-step) (be such as perfused, coat bioreactor, load cell), wherein such step is provided and is only used for Illustration purpose.

It (" grows and receives in hollow-fiber bioreactor system in U.S. Patent Application Serial No.13/269,323 Obtain the configurable method and system of cell ", on October 7th, 2011 submits) and U.S. Patent Application Serial No.13/269,351 (" customizable method and system that cell is grown and harvested in hollow-fiber bioreactor system ", on October 7th, 2011 mentions Hand over) in provide for the task of cell expansion system and the example of scheme and further describe, including customized task and prelist Journey task, these applications are incorporated herein by reference in their entirety herein, for its instructed and for all purposes.

Next, Figure 10 shows the example that can realize the computer system 1000 of embodiment of the present invention on it Component.For example, using processor in cell expansion system to execute task (as such as above process 700,800 and 900 The customized task or pre-programmed mission that a part of process executes) in the case where, computer system 1000 can be used in embodiment party In case.In embodiments, the task of pre-programmed may include, such as, it then follows " IC/EC flushing " and/or " supporting cell ".

As understood by those skilled in the art, computer system 1000 may include user interface 1002, processing system 1004 and/or reservoir (storage) 1006.User interface 1002 may include output equipment 1008 and/or input equipment 1010.Output equipment 1008 may include one or more touch screens, wherein touch screen may include for provide one or The display area of more application widgets.Touch screen can also be input equipment 1010, can be for example from user or operator Receive and/or capture physics touch event.Touch screen may include the liquid crystal display (LCD) with capacitance structure, the capacitor Structure permission processing system 1004 is inferred to the position of touch event, as understood by those skilled in the art.Then, processing system The position of touch event can be mapped to the UI element presented in the predetermined position of application widget by system 1004.According to embodiment party Case, touch screen can also receive touch event by other one or more electronic structures.Other output equipments 1008 can be with Including printer, loudspeaker etc..As understood by those skilled in the art, other input equipments 1010 may include keyboard, its His touch input device, mouse, voice-input device etc..

According to an embodiment of the invention, processing system 1004 may include processing unit 1012 and/or memory 1014. Processing unit 1012, which can be, can operate to execute the general processor for the instruction being stored in memory 1014.According to embodiment party Case, processing unit 1012 may include single processor or multiple processors.In addition, in embodiments, each processor can To be the multi-core processor with one or more cores, to read and execute individual instruction.Such as those skilled in the art Understood, processor may include general processor, specific integrated circuit (ASIC), field programmable gate array (FPGA), Other integrated circuits etc..

According to embodiment, memory 1014 may include for any short of data and/or processor-executable instruction Phase or long-term storage.As understood by those skilled in the art, memory 1014 may include such as random access memory (RAM), read-only memory (ROM) or electrically erasable programmable read-only memory (EEPROM).As those skilled in the art manage Solution, other medium for storing may include such as CD-ROM, tape, digital versatile disc (DVD) or other optical storages, magnetic Band, magnetic disk storage, tape (magnetic tape), other magnetic bunkerages etc..

Reservoir 1006 can be any long term data bunkerage or component.According to embodiment, reservoir 1006 can To include one or more systems described in conjunction with memory 1014.Reservoir 1006 can be permanent or moveable. In embodiments, reservoir 1006 stores the data for being generated or being provided by processing system 1004.

Embodiment

Certain methods/process/scheme/configuration the embodiment for the aspect that embodiment may be implemented is described below.Although Special characteristic can be described in these examples, but is to provide it and is merely to illustrate and describes purpose.The present invention is not limited to following The embodiment of offer.

Embodiment 1

It is shown by implementing the systems and methods amplifying cells and collecting the sample result of extracellular particle (such as EV) In the chart 1200 of Figure 120 0.Chart 1200 show using for example according to the above method of embodiment 700,800 and/or A series of 1204 EV products that 900 and CES 500,600 is run.For example, this system production/collection can be used by section The Terumo BCT company manufacture of the more state Lakewoods of rollerCell expansion system (System or).As shown, the above process and system generation and the EV for collecting various concentration can be used.Production run The EV that can produce various concentration, from the high level 1208 more than 2.5E+07 EV/mL to 5.00E+06 EV/mL and 1.00E+07 Low value 1212 between EV/mL.

Embodiment 2

According to embodiment, generated using the above method 700,800 and/or 900 and/or using system 500,600 and/or Collect extracellular particle (such as EV) sample result be shown in Figure 13 A, 13B and/or 13C chart 1300,1304 and/or In 1308.Each concentration of the EV 1312a and/or 1312c that can be generated by system 500,600 are close to or are greater than in 225cm² The concentration of the EV 1316a and/or 1316c that generate in tissue culture flasks (T225), wherein this bottle can with system 500, 600 similar cell densities inoculations substantially, and be similarly processed (for example, using similar cell density and keeping) with It is compared.As shown in Figure 13 B, in 225cm²The concentration of the EV 1316b generated in tissue culture flasks (T225) can be greater than by System 500,600 generate EV 1312b concentration, wherein this bottle can with the substantially similar inoculation of system 500,600 Density is inoculated with and is similarly processed to be compared.

As an example, Figure 13 A show relative to be inoculated with substantially similar inoculum density and be processed similarly with Those of production and/or collection EV in T-type bottle (" Q1468T- bottles ") 1316a being compared expand system in Quantum cell The possible outcome of EV is generated and/or collected in the operation that the number of system is " Q1468 " 1312a.For example, for Q1468'sSystem (surface area 21,000cm²) bioreactor can load the cell of 1.25E+08 culture, and Q1468T Type bottle (surface area 225cm²) cell that 9.24E+05 is cultivated can be loaded, may be equal to loading has substantially similar cell The Quantum system and T-type bottle of density.Although example results in figure 13a show 1312a (System) and The concentration of 1316a (T-type bottle) between 6.00E+07EV/mL and 7.00E+07EV/mL, the display of chart 1300 with from T-type bottle The concentration for the EV 1316a that (Q1468T type bottle) is collected is compared, fromThe EV1312a concentration that system is collected is higher.

For example, in Figure 13 A and 13C example results can show relative in conventional T-type bottle use similar cell carry The concentration measuring (for example, similar cell density) and keeping amount and obtaining, by system production/collection (for example, using by section sieve The Terumo BCT company of more state Lakewoods is drawn to manufactureCell expansion system) obtain EV concentration it is higher.Make With CES 500,600 may due to several functions possible automation and labor intensity is smaller, and due to bigger surface Product next life long cell, therefore greater number of EV can be provided in some embodiments.

Embodiment 3

According to embodiment, the use-case such as above method 700,800 and/or 900 and/or with system 500,600 generate and/or The sample result for collecting the outer particle (such as EV) of antigen-specific cellular is shown in Figure 14.It as shown in figure 14, can be normal from being obtained from Advise T-type bottle and such as by the Terumo BCT company manufacture of state of Colorado LakewoodCell expansion system System 500,600 purifying excretion body representative sample in obtain sample result.As shown in Figure 140 0, antigentic specificity The type of excretion body may include CD9, CD63 and/or CD81.The many CD9 excretion bodies for generating and collecting from system 500,600 can To be shown in column 1404；The many CD63 excretion bodies for generating and collecting from system 500,600 can be shown in column 1408；From Many CD81 excretion bodies that system 500,600 is generated and collected can be shown in column 1412.Further, cell can with be System 500,600 (for example,System) substantially similar cell density is seeded in 225cm²Tissue culture flasks (T225) In, and be processed similarly to be compared.As shown in chart 1400, from many tissue culture flasks (T225) generation and collected CD9 excretion body can be shown in column 1416；The many CD63 excretion bodies for generating and collecting from tissue culture flasks (T225) can be in column It is shown in 1420；The many CD81 excretion bodies for generating and collecting from tissue culture flasks (T225) can be shown in column 1424.Such as figure Shown in 14, system 500,600 can be higher than tissue culture flasks for the quantity for the excretion body that every kind of antigen is generated and collected (T225) for the quantity for the excretion body that every kind of antigen generates and collects.For example, according to an embodiment, Figure 14 shows considerable The amount for observing the amount ratio T225 cutting of every kind of antigentic specificity excretion body from CES cutting is 2-3 times high.

Embodiment 4

As explained about Fig. 8, can be perfused with PBSSystem (such as CES 500 and/or 600), and Overnight with 5mg FN coating.Second day, system can undergo 2.5X IC EC to rinse and (can have with complete medium GlutaMAX adds the α MEM of 10%FBS) it adjusts.Pre-selected MSC can be inoculated into bioreactor and expand 4-5 It.At this point, system can be carried out with PBS, 5X IC EC is rinsed and negative ultrafiltration is rinsed, to remove as far as possible from bioreactor More serum.Then it can carry out being rinsed (only with the α MEM of GlutaMAX) with the 2.5X IC EC of basal medium with supplement The metabolin lost during PBS is rinsed.Basal medium can be used for supplementing cell 48 (48) hour, while can be by condition Culture medium is collected into harvest bag.Can also with inoculum density inoculation bottle substantially similar with bioreactor and withSystem is similarly handled with for comparative purposes.

For example, expanding six (6) days due to 9,900,000 loading cells bioreactors 501,601, the serum in system is rushed It washes out, excretion body collects two (2) days in the circuit IC, it can be observed that using above-mentioned side from a part as embodiment 4 Method 700,800 and/or 900 and/or system 500,600, to generate 4.71x10¹¹The possible outcome of total excretion body.Collecting excretion After body, excretion body can be harvested from the circuit IC, wherein it can be observed that having recycled 1.35 hundred million from bioreactor 501,601 MSC。

Embodiment of the present invention can have one or more aspects, including for example:

Embodiment of the present invention and/or aspect may include the method for collecting cellular products, this method comprises: cell is filled It is downloaded in bioreactor；Cell is supported with culture medium；Amplifying cells, wherein cell discharges cellular products；Concentration discharges thin Born of the same parents' product；And the cellular products of concentration are collected from bioreactor.

Any of one or more the embodiment above and/or aspect, wherein the cellular products through discharging include thin Extracellular particle.

Any of one or more the embodiment above and/or aspect, wherein extracellular particle includes cell external capsule Bubble.

Any of one or more the embodiment above and/or aspect, wherein extracellular vesica includes excretion body.

Any of one or more above embodiment and/or aspect, wherein extracellular vesica includes microcapsule bubble.

Any of one or more the embodiment above and/or aspect, wherein extracellular particle includes that virus carries Body.

Any of one or more the embodiment above and/or aspect, wherein collect the cellular products through harvesting Into bag.

Any of one or more above embodiment and/or aspect further comprise: replacement culture medium.

Any of one or more the embodiment above and/or aspect, wherein culture medium includes serum.

Embodiment of the present invention and/or aspect may include cell expansion system, it includes bioreactor, wherein institute Stating bioreactor includes hollow-fibre membrane；First fluid flow path has at least opposite end, wherein first fluid stream Dynamic path is connected with the capillary inner part fluid of hollow-fibre membrane；Processor；Memory, with processor communication and can be by It manages device to read, and includes series of instructions, when being executed by a processor, so that processor: receiving selection to support cell；It connects The outlet of capillary inner part is closed by selection, wherein closing the outlet of capillary inner part makes from capillary inner part The particulate condensation of cell release；And it is operated so that particle to be moved in harvest bag.

Any of one or more the embodiment above and/or aspect further comprise one of the following or more It is a: to be operated to execute 5x flushing；It is operated to execute negative ultrafiltration；And/or it is operated to execute flushing.

Any of one or more the embodiment above and/or aspect, wherein the particle discharged from cell includes thin Extracellular particle.

Any of one or more the embodiment above and/or aspect, wherein extracellular particle includes excretion body.

Any of one or more the embodiment above and/or aspect, wherein extracellular particle includes microcapsule bubble.

Any of one or more the embodiment above and/or aspect further comprise: receiving and select in replace Fluid in the capillary inner part and extracapillary part of empty fiber membrane.

Any of one or more the embodiment above and/or aspect, wherein the replacement of the fluid is from for supplying It supports in the first culture medium of cell and extracts protein.

Any of one or more the embodiment above and/or aspect, wherein nonprotein second culture medium Replace the first culture medium to support cell.

Any of one or more the embodiment above and/or aspect further comprise being operated in life The test of the first and/or second culture medium is executed in object reactor to determine whether protein has been removed.

Embodiment of the present invention and/or aspect may include cell expansion system, include: bioreactor, wherein described Bioreactor includes hollow-fibre membrane；First fluid flow path has the at at least opposite both ends of bioreactor One entrance and first outlet, wherein first fluid flow path is connected with the capillary inner part fluid of hollow-fibre membrane；Second Fluid flow path has second entrance and second outlet, wherein the capillary in second fluid flowing path and hollow-fibre membrane Outer portion fluid is connected；First connectivity port is connected with first fluid flow path fluid, wherein being attached to the first connectivity port First bag introduce cells into bioreactor；Second connection end mouth is connected, wherein wrapping with first fluid flow path fluid Second bag containing the first culture medium is connected to second connection end mouth, to be provided the first culture medium by first fluid flow path To bioreactor, to support cell to double until the cell of pre-determined number has occurred, first culture medium contains protein； Third connectivity port is connected with first fluid flow path and second fluid path fluid, wherein comprising the second culture medium Third bag is connected to third connectivity port, the second culture medium is supplied to bioreactor by first fluid flow path, And second connection end mouth provides the second culture medium by first fluid flow path.Fluid path is rinsed from bioreactor First culture medium out；4th connectivity port is connected with second fluid flowing path fluid, wherein includes the after being flushed The 4th bag of three culture mediums is connected to the 4th connectivity port to support cell, wherein when supporting cell with third culture medium, the The first outlet of one fluid flow path is closed, with for the cell release being concentrated from the capillary inner part of bioreactor Grain, the third culture medium are free of protein；Harvest bag is connected with first fluid flow path fluid, and wherein concentrated granular is mobile Into harvest bag.

Embodiment of the present invention and/or aspect may include the side for generating cell granulations in cell expansion system Method, this method comprises: perfusion cell expansion system, wherein cell expansion system includes: bioreactor, wherein bioreactor Include: hollow-fibre membrane, there is capillary inner part and extracapillary part；First fluid flow path, in bioreactor At least opposite both ends there is first entrance and first outlet, the wherein capillary of first fluid flow path and hollow-fibre membrane Pipe inner part fluid is connected；Second fluid flows path, has second entrance and second outlet, and wherein second fluid flows path It is connected with the extracapillary segment fluid flow of hollow-fibre membrane；First connectivity port is connected with first fluid flow path fluid；The Two connectivity ports are connected with first fluid flow path fluid；Third connectivity port, with first fluid flow path and second Body path fluid is connected；4th connectivity port is connected with second fluid flowing path fluid；And harvest bag；First bag is connected To the first connectivity port to introduce cells into bioreactor；Second bag comprising the first culture medium is connected to the second connection Mouthful, the first culture medium is supplied to bioreactor by first fluid flow path, to support cell until occurring predetermined The cell of number doubles, and first culture medium contains protein；After the cell multiplication for having occurred and that pre-determined number, it will wrap Third bag containing the second culture medium is connected to third connectivity port, will to pass through first fluid flow path and second fluid path Second culture medium is supplied to bioreactor, to flush out the first culture medium from bioreactor；After being flushed, the is closed The first outlet of one fluid flow path, with the particle for the cell release being concentrated from the capillary inner part of bioreactor； The 4th bag comprising third culture medium is connected to the 4th connector to support cell, the third culture medium is free of protein； Harvest bag is connected to first fluid flow path to harvest；And by the particle of concentration harvest into harvest bag

Embodiment of the present invention and/or aspect may include in one or more the embodiment above and/or aspect The combination of any one.

Embodiment of the present invention and/or aspect may include for executing one or more above-described embodiments and/or side The method in any one of face.

Although having illustrated and described example embodiment and application of the invention, it should be appreciated that, the present invention is not It is limited to above-mentioned accurate configuration and resource.It without departing from the scope of the invention, can be in present invention disclosed herein Various modifications, change and the change that will be apparent to those skilled in the art in the arrangement of method and system, operation and details Change.

As used herein, " at least one (kind) ", " one (kind) or more (kind) " and "and/or" are both to have covered knot Meaning when sharing covers the open language of the meaning of separated used time again.For example, expression " at least one of A, B and C (kind) ", " at least one of A, B or C (kind) ", " one (kind) in A, B and C or more (kind) ", " one of A, B or C (kind) or more (kind) " and " A, B and/or C " indicate only A, only B, only C, A and B together, A and C together, B and C together or A, B and C are together.

It will be apparent to one skilled in the art that without departing from the scope of the invention, it can be to this The method and structure of invention carry out various modifications and changes.It should therefore be understood that the specific example that the present invention is not limited to provide. On the contrary, the present invention is directed to cover the modifications and variations in the range of the following claims and their equivalents.

Claims

1. a kind of method for collecting cellular products, which comprises

By loading cells into bioreactor；

The cell is supported with culture medium；

Expand the cell, wherein the cell discharges cellular products；

The cellular products through discharging are concentrated；And

Concentrated cellular products are harvested from the bioreactor.

2. method described in claim 1, wherein the cellular products through discharging include extracellular particle.

3. method as claimed in claim 2, wherein the extracellular particle includes extracellular vesica.

4. method as claimed in claim 3, wherein the extracellular vesica includes excretion body.

5. method as claimed in claim 3, wherein the extracellular vesica includes microcapsule bubble.

6. method as claimed in claim 2, wherein the extracellular particle includes viral vectors.

7. method described in claim 1, wherein the cellular products through harvesting are collected into bag.

8. method described in claim 1, further comprises:

Replace the culture medium.

9. method described in claim 1, wherein the culture medium includes serum.

10. a kind of cell expansion system, comprising:

Bioreactor, wherein the bioreactor includes hollow-fibre membrane；

First fluid flow path, have at least opposite end, wherein the first fluid flow path with it is described hollow The capillary inner part fluid of tunica fibrosa is connected；

Processor；

Memory, the memory and the processor communication and can be read by the processor, and include a series of fingers It enables, when being executed by the processor, so that the processor:

Selection is received to support cell；

Selection is received to close the outlet of the capillary inner part, wherein the outlet for closing capillary inner part makes from described The particulate condensation of cell release in capillary inner part；And

It is operated so that the particle to be moved in harvest bag.

11. cell expansion system described in any one of claim 10 further comprises the one or more of following item:

It is operated to execute 5x flushing；

It is operated to execute negative ultrafiltration；And/or

It is operated to execute flushing.

12. cell expansion system described in any one of claim 10, wherein the particle discharged from the cell includes extracellular Grain.

13. cell expansion system described in claim 12, wherein the extracellular particle includes excretion body.

14. cell expansion system described in claim 12, wherein the extracellular particle includes microcapsule bubble.

15. cell expansion system described in any one of claim 10, further comprises:

Selection is received to replace the fluid in the capillary inner part of the hollow-fibre membrane and extracapillary part.

16. cell expansion system described in claim 15, wherein the replacement of the fluid is from for supporting the of the cell Protein is extracted in one culture medium.

17. cell expansion system described in claim 16, wherein nonprotein second culture medium replacement, first training Base is supported to support the cell.

18. cell expansion system described in claim 17, further comprises:

It is operated to execute the test of first culture medium and/or the second culture medium in the bioreactor with determination Whether the protein has been removed.

19. a kind of cell expansion system, comprising:

Bioreactor, wherein the bioreactor includes hollow-fibre membrane；

First fluid flow path at at least opposite both ends of the bioreactor there is first entrance and first to go out Mouthful, wherein the first fluid flow path is connected with the capillary inner part fluid of the hollow-fibre membrane；

Second fluid flows path, with second entrance and second outlet, wherein second fluid flowing path with it is described The extracapillary segment fluid flow of hollow-fibre membrane is connected；

First connectivity port is connected with the first fluid flow path fluid, wherein is attached to first connectivity port First bag introduce cells into the bioreactor；

Second connection end mouth is connected comprising second bag of the first culture medium with the first fluid flow path fluid It is connected to the second connection end mouth, first culture medium is supplied to the life by the first fluid flow path Object reactor, to support the cell to double until the cell of pre-determined number has occurred, first culture medium contains protein；

Third connectivity port, be connected with the first fluid flow path and the second fluid path fluid comprising The third bag of second culture medium is connected to the third connectivity port, to pass through the first fluid flow path and described second Second culture medium is supplied to the bioreactor by fluid path, to flush out described from the bioreactor One culture medium；

4th connectivity port is connected with second fluid flowing path fluid, wherein includes the after the flushing The 4th bag of three culture mediums is connected to the 4th connectivity port to support the cell, wherein when with the third culture medium When supporting cell, the first outlet of the first fluid flow path is closed so that the capillary from the bioreactor is concentrated The particle of cell release in pipe inner part, the third culture medium are free of protein；And

Harvest bag is connected, wherein concentrated particle is moved to the harvest bag with the first fluid flow path fluid In.

20. a kind of method for generating cell granulations in cell expansion system, which comprises

The cell expansion system is perfused, wherein the cell expansion system includes:

Bioreactor, wherein the bioreactor includes:

Hollow-fibre membrane, the hollow-fibre membrane have capillary inner part and extracapillary part；

First connectivity port is connected with the first fluid flow path fluid；

Second connection end mouth is connected with the first fluid flow path fluid；

Third connectivity port is connected with the first fluid flow path and the second fluid path fluid；

4th connectivity port is connected with second fluid flowing path fluid；And

Harvest bag；

First connectivity port is connected to by first bag to introduce cells into the bioreactor；

Second bag comprising the first culture medium is connected to the second connection end mouth, to pass through the first fluid flow path First culture medium is supplied to the bioreactor, to support the cell until the cell times of pre-determined number occurs Increase, first culture medium contains protein；

After the cell multiplication for having occurred and that the pre-determined number, the third bag comprising the second culture medium is connected to described the Three connectivity ports, to be supplied to second culture medium by the first fluid flow path and the second fluid path Bioreactor, to flush out first culture medium from the bioreactor；

After the flushing, the first outlet of the first fluid flow path is closed, to be concentrated from the bioreactor Capillary inner part in the cell release particle；

The 4th bag comprising third culture medium is connected to the 4th connectivity port to support the cell, the third culture Base is free of protein；

The harvest bag is connected to the first fluid flow path to harvest；And

Concentrated particle is harvested into the harvest bag.